Identification and production of novel potential pathogen-specific biomarkers for diagnosis of histoplasmosis

ABSTRACT Immunological assays based on the detection of circulating fungal biomarkers are helpful in clinical practice for diagnosing invasive fungal infections. Some of these targeting antigenic components are common to several different fungi. Histoplasmosis is a mycosis caused by the dimorphic fungus Histoplasma capsulatum, which in recent years has gained significant relevance due to the increase in the population susceptible to developing severe clinical forms, including those living with HIV/AIDS. Immunological tests that detect cell wall polysaccharide antigens are among the most used laboratory techniques for diagnosing this mycosis. However, none have shown adequate performance, and cross-reactivity with other fungal pathogens may be observed. Considering that there is a real need to improve the sensitivity and specificity of current diagnostic methods, we explored a novel strategy for the identification of H. capsulatum-specific antigens (Hc_Ags) that could be detected in clinical samples during infection based on a computational analysis model that included the application of bioinformatics tools and the analysis of experimental data from transcriptomics and proteomics. The Hc_Ags identified were expressed and purified by eukaryotic and prokaryotic systems. First, the Hc_Ags were used in an in-house immunization model in mice (BALB/c) to obtain Hc_Ag-specific polyclonal antibodies (Hc_Ag_PAb). Then, the presence of these antigens in H. capsulatum-yeast culture extracts and the specificity of Hc_Ag_PAb against culture extracts of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Fusarium spp., and Paracoccidioides brasiliensis were confirmed. Finally, we demonstrated the immunoreactivity of these Hc_Ag-specific polyclonal antibodies with urine samples from patients previously diagnosed with histoplasmosis. IMPORTANCE Histoplasmosis is considered one of the most important mycoses due to the increasing number of individuals susceptible to develop severe clinical forms, particularly those with HIV/AIDS or receiving immunosuppressive biological therapies, the high mortality rates reported when antifungal treatment is not initiated in a timely manner, and the limitations of conventional diagnostic methods. In this context, there is a clear need to improve the capacity of diagnostic tools to specifically detect the fungal pathogen, regardless of the patient’s clinical condition or the presence of other co-infections. The proposed novel pathogen-specific biomarkers have the potential to be used in immunodiagnostic platforms and antifungal treatment monitoring in histoplasmosis. In addition, the bioinformatics strategy used in this study could be applied to identify potential diagnostic biomarkers in other models of fungal infection of public health importance.


Analysis of orthologous proteins
A comparative analysis of 343,723 proteins obtained from 33 proteomes of fungi species and 2 proteomes of Mycobacterium tuberculosis was performed using the OrthoMCL algorithm (Table S1).Of the 25,770 OrthoMCL protein clusters obtained, 1,851 (7%) contained at least one protein from each species included in the analysis.Not surpris ingly, these proteins are involved in central biological processes, including cell cycle regulation, transport, cytoskeleton organization, or as surface antigens (data not shown).Further exploration of the data sets showed that most clusters contained between two and nine proteins (n: 19,618 clusters, 76%), and only a group of five clusters included more than 1,000 predicted orthologs genes (Table 1).For each fungal species, the clusters with at least one protein, the clusters with in-paralogs proteins (genes present in a particular fungal species that are related to each other through a gene duplication event, without orthologs genes found in different species), and the total number of singleton proteins (genes for which no orthologous or paralogous relationship was found) are presented in Fig. 1.Interestingly, H. capsulatum presented a group of 1,567 protein clusters containing a total of 3,572 in-paralogs proteins and 3,230 singletons proteins, which were determined as specific to this fungus by OrthoMCL.A total of 6,802 Hc-proteins were selected for the subsequent analysis.

Identification of potential biomarkers
We designed a strategy to find those proteins specific to H. capsulatum that can serve as potential biomarkers for diagnosis.The overall analysis pipeline is illustrated in Fig. 2.
Following OrthoMCL analysis, the next step was to use TargetP and SignalP to detect H. capsulatum proteins that contain a signal peptide (SP) in the N-terminal region, which is responsible for exporting proteins to extracellular environment.Thus, if a gene had this signal, it was considered a secretory protein.SignalP output revealed 176 genes (101 in-paralogs and 75 singletons) predicted to be classically secreted proteins.In contrast, TargetP predicted 108 proteins that contained secretory pathway signal peptides (56 in-paralogs and 52 singletons).The differences between the two programs could be that SignalP predicts the presence of N-terminal (and cleavable) signal peptides, while TargetP includes the prediction of transmembrane segments associated with other subcellular localizations (cytoplasmic space or cellular membrane) (36).Only proteins predicted by both programs were selected to confirm the analysis further.A total of 57 Hc-proteins were identified as putative secreted proteins (34 in-paralogs and 23 singletons).(Fig. 2).Then, BLASTp against NCBI non-redundant (nr) database was used to rule out proteins that shared similarities with proteins from other species that were not included in the initial OrthoMCL analysis.At last, 54 proteins (31 in-paralogs and 23 singletons) were considered specific to H. capsulatum (Fig. 2).
As a final step, we analyzed previously published experimental data sets and combined them with our orthological analysis to identify potential biomarkers for diagnosing histoplasmosis (Fig. 2).At first, the previously published H. capsulatum extracellular proteome database was used (33).Unfortunately, all proteins described as the core set of extracellular proteins produced by this fungus were found in clusters with similar proteins from other fungi and were initially discarded within the analysis.We also used a transcriptome database with the phase-specific gene profiles (yeast or mycelium) of H. capsulatum previously published by Edwards et al. (34).Interestingly, two genes encoding proteins previously defined in our study as H. capsulatum-specific were identified within a subset of differentially expressed genes.One of them showed significantly enriched expression (50-fold) in the yeast phase compared to the mycelia phase (Hc110_Ag), and the other was a yeast-phase-specific gene (Hc155_Ag).Both genes were considered potential candidates for biomarker diagnosis (Table 2).
Furthermore, a urine-peptides database from immunoassay-positive patients with disseminated histoplasmosis previously published by Crockett et al. was used (35).Considering that this report was able to identify several Histoplasma proteins, which are not necessarily secreted but represent the urine-proteome associated with Histo plasma-antigenuria, a comparative analysis was performed with the total all-Hc-proteins identified by OrthoMCL plus an nr_BLASTp (NCBI database) analysis.The results showed that three urine peptides were closely matched with two Hc-specific proteins according to our analysis (Hc670_Ag and Hc724_Ag), and both proteins were considered diagnostic biomarkers of histoplasmosis and selected for the subsequent experimental process (Table 2).Unsurprisingly, most of the peptides published in that study corresponded to proteins that shared homology with proteins from other fungi (Table S2).Due to this, all protein clusters obtained by OrthoMCL paired with any urine peptides of H. capsu latum were further analyzed.Finally, we found that one of these clusters, related to two urine peptides, contained 16 homologs proteins (according to OrthoMCL) from the fungal genera; Paracoccidioides (3), Blastomyces (2), Emmonsia (2), Cryptococcus (2), and seven in-paralogs H. capsulatum, designed at Hc212_Ag.A subsequent BLASTp analysis revealed that the percent match and query coverage were <40% between those proteins.Considering that an antigenic epitope can be defined as a short sequence of 10 to 16 amino acids (linear and/or conformational), the low sequence similarity observed between the proteins of the cluster, and the size of the identified protein (212 aa), this Hc-protein (Hc212_Ag) was considered a potential diagnostic biomarker (Table 2).

Protein expression and immunoreactivity
Initially, the construct of the expression vector (pET-100D/TOPO plasmid) for each candidate was obtained by GeneArt (Gen Synthesis Services, USA).All candidates for diagnostic biomarkers, except for Hc212_Ag, were expressed by a prokaryotic recombi nant protein expression system, induced with IPTG in Escherichia coli BL21 (strain DE3) and purified by affinity column chromatography (as described in Materials and Methods).The Hc212_Ag was obtained by external service of recombinant protein expression in eukaryotic cells (GenScript, Biotech Corporation, USA) since it presented post-transla tional glycosylation-type modifications.The expression of purified recombinant proteins, Hc670_Ag and Hc212_Ag, was confirmed by SDS-PAGE, and the molecular weights were found to be around 75 kDa and 40 kDa, respectively (Fig. 3A and C).Hc110_Ag, Hc155_Ag, and Hc724_Ag were not possible to express and purify at the time of this publication, so they will be used in other future studies.
The SDS-PAGE of purified proteins and culture extracts of H. capsulatum (yeast), Candida albicans (yeast), Aspergillus fumigatus (mycelium), Cryptococcus neoformans (yeast), Paracoccidioides brasiliensis (yeast), and Fusarium spp.(mycelium) were immu noblotted with immunized mice's sera and the reactivity of the PAbs anti-Hc670_Ag and anti-Hc212_Ag was observed only with H. capsulatum proteins (Hc212_Ag and Hc670_Ag) and H. capsulatum yeast extracts (Fig. 3B and D) but not with the other fungal extracts (Fig. 4B and C), except for P. brasiliensis, which showed multiple reactiv ity bands with both PAbs, anti-Hc670_Ag and anti-Hc212_Ag.As a control, commercial monoclonal antibody anti-C.albicans (Fig. 4D) and serum anti-Aspergillus from patients with confirmed aspergillosis invasive (Fig. 4E) were used against fungal extracts.Finally, preliminary experiments were performed with anonymized urine samples from patients with confirmed disseminated histoplasmosis by IMMY ALPHA EIA test.It was observed that both anti-Hc670_Ag and anti-Hc212_Ag were immunoreactive with the samples (Fig. 5A and B), indicating that both specific H. capsulatum proteins have potential use as diagnostic biomarkers for human histoplasmosis.

DISCUSSION
In fungal infections, the concept of circulating antigens in infected host serum as potential biomarkers of disease has been reported for many years (37)(38)(39)(40).Currently, immunoassay tests involving fungal antigen detection are widely used in clinical practice to diagnose and monitor antifungal treatment.
In the case of histoplasmosis, the detection of circulating antigen has been descri bed as the best option for diagnosis, mainly in immunocompromised patients with disseminated histoplasmosis (9,41).There are two major EIA tests based on the detection of low molecular weight HPA present in the urine and serum of patients, which have been validated in two laboratories: Mvista Histoplasma Quantitative EIA test (Miravista Diagnostics, Indianapolis, USA) and IMMY ALPHA Histoplasma Antigen EIA test (Immuno-Mycologics, Inv., Norman, OK) (42).Nevertheless, several authors have reported discrepant results with samples analyzed with tests and cross-reactions with other fungal infections (11,14,(43)(44)(45).Recently, one of these companies developed a new lateral flow assay for detecting HPA in urine samples (46,47) that may allow a point-of-care diagnosis of histoplasmosis.However, cross-reactivity is still one of the significant challenges.Likewise, it has been described that other antigens of H. capsulatum commonly used in serodiagnosis, such as M and H antigens, exhibit cross-reactivity with other dimorphic fungi (48,49).Considering that currently no specific and clearly defined biomarkers of H. capsulatum are known, we used several analytical approaches to identify potential biomarkers for the diagnosis of histoplasmosis by applying different bioinformatics tools, including computational platforms for prediction of orthologs and secretory proteins  Indeed, the recent advances in next-generation sequencing technologies, the increasing genome and protein entries into NCBI (National Center of Biotechnology Information), and the development of new bioinformatics algorithms have facilitated comparative genomics and proteomics studies.The OrthoMCL is an algorithm widely used for identifying orthologs of proteins between multiple species based on sequence similarity (all vs all BLASTp), which also allows recognition of those specific proteins for each species (27,(50)(51)(52).Our study used a predicted proteome of four H. capsulatum strains (G186A, H88, H143, and Nam1).These proteomes exhibited differences in genome length (Mbp) and the number of annotated proteins that could be related to a non-iden tical workflow of sequencing and assembly defined by each NCBI-Bioproject per strain (53).
By OrthoMCL analysis, the H. capsulatum proteomes were compared with other proteomes of clinically significant or phylogenetically related fungal pathogens that also infect immunocompromised patients and cause nonspecific and indistinguishable symptoms (8,15,54).Additionally, despite the noticeable and recognized difference between fungi and bacteria, the Mycobacterium tuberculosis proteome was included in the analysis, considering the high incidence of histoplasmosis with pulmonary tuberculo sis cases reported among people living with HIV/AIDS and the similarity of the clinical and radiological presentations between both (55,56).The results obtained showed that 1,851 of the 25,770 protein groups formed by OrthoMCL contained proteins from each fungus species with high genetic similarities and were considered homologs proteins, which could be a consequence of common ancestry, horizontal transfer in a shared habitat, or a mixture of both (57,58).Additionally, the present analysis showed that for each fungus species, a group of unique proteins called "singletons" could be used in future studies focused on identifying new diagnostic biomarkers.
Particularly, H. capsulatum showed a total of 3,230 singleton proteins and multiple in-paralogs protein clusters with high sequence similarities (1,567 clusters for a total of 3,572 proteins).However, each protein in these paralogs clusters was evaluated individually.Alternately, a BLASTp analysis using NCBI-compiled nr databases contain ing non-redundant RefSeq protein records from GenBank and other protein data bank archives (PDB, Swiss-Prot, UniProt, etc.) was considered in our analysis, which improved the precision of the study by allowing the identification of particular H. capsulatum proteins (59,60).
Our strategy also included the prediction of putative secreted proteins that contained the signal sequence and were identified as targeting the classical secretory pathway.All Hc-proteins were analyzed with the amino-acid sequence-based predictors SignalP and TargetP.Thus, only those proteins that had the following characteristics: (i) an N-terminal SP; (ii) no transmembrane domains; and (iii) no predicted localization signal to other intracellular organelles, were considered "secreted Hc-proteins."Further analysis of non-classical pathways was not considered since few proteins secreted by this pathway have been described in fungi and the current bioinformatics programs focused on this approach, such as SecretomeP 2.0, are not fully ported and only work for mammalian and Gram-negative proteins (61,62).Overall, this prediction approach would allow us to target possible circulating antigens the fungus releases during an infection.
Even with the robustness and extensive use of computational biology, it is essential to validate the scientific finding through empirically based knowledge or experimental methods that enhance the confidence and accuracy of predictive computational models (63,64).Accordingly, we used a previously published, unique, and representative data set of peptides obtained directly from urine samples of patients with disseminated histoplasmosis (35,65).In this study, the authors reported that some peptides contained sequences homologous to conserved hypotheticals H. capsulatum proteins.However, the specificity of these peptide-related proteins was not fully verified.Interestingly, through this data set, we identified three proteins, two of them considered by OrthoMCL as singletons proteins of H. capsulatum (Hc670_Ag and Hc724_Ag), that would have experimental evidence as circulating antigens in the urine (and presumably in the serum) of infected patients.
Both Hc670_Ag and Hc724_Ag are classified as predicted proteins with unknown biological function, molecular or biochemical structure, or recognized orthologs or homologs (based on sequence similarity).Several reports have described that approx imately 80% of H. capsulatum yeast-phase-regulated genes encode hypothetical and uncharacterized unknown proteins, highlighting how little is currently understood about the biology of H. capsulatum and the need for further studies focused on characterizing at the molecular level and gaining a comprehensive view of genes that can serve as potential biomarkers for diagnosis (33,34,66,67).Coincidentally, some authors have reported the production of murine monoclonal antibodies with potential use in epidemiology and serodiagnosis that recognized an H. capsulatum antigen with an apparent molecular mass of 70-75 kDa, like Hc670_Ag, but exhibited weak reactivity to antigens derived from Sporothrix, Paracoccidioides, or Blastomyces (68)(69)(70).However, these studies utilized a whole yeast cell extract that could contain antigens shared with other fungal pathogens, contrary to the use of individual H. capsulatum-specific antigens as proposed in this study.
Regarding the other identified protein, Hc212_Ag, it is a predicted glycosylated protein that was matched with a secreted and yeast-phase-specific protein with unknown functions previously designated as Cfp-4 (Culture filtrate protein) (33), and linked to an ortholog group of proteins from H. capsulatum and other fungi.How ever, some reports have described that OrthoMCL can cluster genes with high-scoring (bit-score) alignments that are not homologs or share functional similarities (59,71,72).Therefore, by analyzing all protein clusters paired with any of the H. capsulatum urine peptides, we found that, particularly, a protein cluster with seven in-paralogs proteins of H. capsulatum (homologs to Cfp-4) and nine putatively homologs proteins from other fungi [Paracoccidioides (3), Blastomyces (2), Emmonsia (2), and Cryptococcus (2)], unexpectedly had a low sequence similarity.The above, added to the fact that Cfp-4 was described as one of the main extracellular factors produced by H. capsulatum, although it does no apparent role in the virulence or pathogenesis of infection (33,73), could be considered as an H. capsulatum-specific yeast-phase exoantigen.More recently, Gallo et al. reported the design of primer sets for two genes, PPK (predicted protein kinase) and Cfp-4, to conventional and real-time PCR assays for the identification of H. capsulatum isolates using purified DNA (74).Similarly, Rubio-Carrasquilla et al. observed that there are no proteins similar to Cfp-4 in any other organism (BLAST search) and identified it as an H. capsulatum-specific immunogenic protein (75).However, to our knowledge, there is no investigation evaluating this protein's potential utility as a diagnostic histoplasmosis biomarker.
Under this same approach, we identified two other proteins (Hc110_Ag and Hc115_Ag) predicted as putative secreted proteins of H. capsulatum and differentially upregulated in the yeast phase (related to active infection) that could have great potential as diagnostic biomarkers.Until now, non-identical or similar proteins have been reported previously.However, as of this publication's date, it was impossible to fine-tune the protein synthesis and purification processes and verify their presence in yeast culture extracts.Thus, these will be analyzed in further studies.
On the other hand, other strategies focused on identifying extracellular antigens released during infection have been published with Candida and Aspergillus.These strategies were based on producing hybridoma cell lines and MAbs from specific structural and metabolic components, such as germ-tube-specific antigens, hyphal cell wall antigens, or galactomannan-like antigens (3,(76)(77)(78).However, lyophilized mycelium or ethanol-precipitated exoantigens from mycelial culture were used as immunogens, and cross-reactivity could be expected since many of these antigens contain epitopes and carbohydrates residues shared with other fungal pathogens, implying that many MAbs must be screened to identify specific individual epitopes.In our case, specific purified proteins were administered with an adjuvant to boost the induction of effective antibody response, which minimizes the risk of cross-reactivity with another fungal pathogen.Likewise, the production of PAbs was chosen, considering our primary purpose, and they are cheaper, easier, and quicker to generate than MAbs, which is very expensive and requires considerable time to produce (6 and 9 months) (79,80).Furthermore, it has been demonstrated that PAb has greater sensitivity and performance if cross-reactivity can be avoided (43).In our case, there was no reactivity with culture extracts of C. albicans, A. fumigatus, C. neoformans, and Fusarium spp., except for P. brasiliensis extracts, which had multiple bands.This could be due to the close relation ship and genetic proximity between H. capsulatum and P. brasiliensis (81)(82)(83), and the potential presence of proteins with some small shared regions that polyclonal antibodies can recognize.However, there are significant differences regarding their geographic distribution, clinical characteristics, patients, and risk factors that determine the correct diagnosis procedures and therapeutic management (84)(85)(86).
Finally, both PAbs (anti-Hc212_Ag and anti-Hc670_Ag) were shown to be reactive against purified antigens, H. capsulatum yeast culture extracts, and samples from patients with a confirmed diagnosis of histoplasmosis.Previously, it has been suggested that most yeast-phase culture filtrates are characterized by a prominent slower mobility smear consistent with the presence of highly glycosylated proteins, including some with low molecular weight, like those observed at the top of the membrane in the western immunoblot (33,70,87,88).This result could be attributed to the polysaccharide nature of Hc212_Ag and other unknown proteins homologs to Hc670_Ag present in H. capsulatum.
Considering that the major impediment to the development of a new specific immunoassay for the diagnosis of infectious disease is the identification of appropriate antigen targets, this strategy has the potential to be used as a platform to identify new diagnostic biomarkers from a broad spectrum of microbial pathogens.The antigens Hc670_Ag and Hc212_Ag specific for H. capsulatum are two potential antigens identified by this strategy and serve as proof of concept.However, future studies are necessary to evaluate the performance of new immunological diagnostic platforms with our anti-Histoplasma PAbs in terms of sensitivity and specificity with samples from patients with histoplasmosis and other fungal infections.

Comparative analysis and ortholog prediction
To identify those proteins specific to H. capsulatum, a simple analysis of orthologs species was determined using the OrthoMCL algorithm (27) with all evaluation parameters established by default, including the following: P value cutoff: 1e −5 , percent identity and percent match cutoffs: 30%, maximum weight: 100, and Markov inflation index: 1.5.
All evaluation parameters of the software were set by default.A total of 343,723 protein sequences were included in a local proteome database obtained from ref erences strains collection of GenBank database, available at the NCBI ftp site (ftp:// ftp.ncbi.nih.gov/).The collection was generated with 35 proteomes from 12 genera of fungi as follows: Histoplasma sp., Emmonsia spp., Blastomyces sp., Paracoccidioides sp., Coccidioides sp., Candida sp., Cryptococcus spp., Pneumocystis sp., Aspergillus sp., Talaromyces sp., Fusarium sp., Schizophyllum sp., and two proteomes of bacterium Mycobacterium tuberculosis (see Table S1).

Prediction of secreted antigenic proteins
Complementary, secretory domain prediction was performed using SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP)(31) to determine the presence and location of signal peptide cleavage sites and the TargetP 2.0 server (https://services.healthtech.dtu.dk/service.php?TargetP-2.0)(30) to predict other subcellular locations by N-terminal pre-sequences (mitochondrial transit peptide, chloroplast transit peptide, or thylakoid luminal transit peptide) and not consider them as secreted proteins.
All proteins identified as Hc-specific by OrthoMCL were processed with both aminoacid sequence-based predictors to establish the presence of signal peptides and their respective subcellular locations (mitochondrial or secretory pathway).Only those proteins with a score value above the threshold in both programs were considered as potentially secreted antigens.

Comparative analysis with experimental data set
Databases from the previously published experimental data set were integrated into the study.The Hc-proteins obtained by orthological analysis were compared with a secreted proteome database obtained from pathogenic yeast-phase H. capsulatum culture filtrates (33), a Histoplasma yeast and mycelial transcriptomes database (34), and a urine-peptides database from Histoplasma-immunoassay-positive patients (35).Only the proteins that matched with any homologs of these experimental databases were considered for further experiments as a candidate for diagnostic biomarker.

Expression and purification of recombinants proteins
For each Hc_Ag gene (Hc110_Ag, Hc115_Ag, Hc670_Ag, and Hc724_Ag), the expression vectors were obtained by GeneArt (Gene Synthesis Services, ThermoFisher Scientific, USA).The services included the full-length gene cloning into pET-100 D/TOPO expression with a polyhistidine (6× His) tag at the N-terminal region to facilitate its purification by affinity binding to a nickel-charged agarose resin (Ni-NTA).E. coli BL21 (DE3) were subsequently transformed and cultured.Expression of the protein in E. coli was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 6 h.Then, cells were harvested by centrifugation at 5,000 rpm for 10 min at 4°C and resuspended with lysis buffer [Phosphate-buffered saline (PBS); 500 mM NaCl; 4% L-sarcosyl], followed by a freeze-thaw cycle with liquid nitrogen and incubation at 37°C for 30 min to solubilize the inclusion bodies that contain the recombinant protein.The cell extracts were centrifuged at 5,000 rpm × 10 min at 4°C to perform the recombinant His-tagged protein purification.The supernatant was passed through a HisPur Ni-NTA Spin column (ThermoFisher Scientific, USA) to bind the His-tagged Hc_Ag protein.Afterward, the column was washed two times with washing buffer (PBS; 500 mM NaCl; 10 mM imidazole), and the bound protein was eluted with elution buffer (PBS; 500 mM NaCl; 100 mM imidazole, pH 6.0).
Purified Hc212_Ag was obtained by GenScript (Protein Expression Services, GenScript Biotech Corporation, New Jersey, USA) through the mammalian Chinese Hamster Ovary expression system.
Expression of both Hc_Ags was confirmed by SDS-PAGE.The concentration was determined for the Bradford protein Assay.The purified recombinant proteins were stored at −80°C.

In vivo immunization model
BALB/c mice were obtained from the breeding colony maintained at Corporación para Investigaciones Biológicas, CIB (Medellín, Colombia).A rapid immunization schedule previously described was adapted to recover polyclonal antisera (89).Briefly, 8-10week-old female BALB/c mice were immunized with purified Hc-proteins following one intraperitoneal (i.p.) injection at day 0 (25 µg of Hc_Ag per injection, mixed in solu tion with an equal volume of Freund's complete adjuvant) and four i.p. injections at days 7, 14, 21, and 28 (25 µg per injection) with Freund's incomplete adjuvant.Three days after the last injection, the total blood volume of immunized mice was collected (polyclonal antisera).Additionally, spleen cells from immunized mice were stored at −80°C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (Gibco, Invitrogen Corporation, California, USA) for further studies.
This study followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and followed the Colombian (Law 84/1989, Resolution no.8430/1993), European Union, and Canadian Council on Animal Care regulations.The protocol was approved by the Institutional Ethics Committee of the CIB.

Fungal culture
Yeast culture of Histoplasma capsulatum (strain G186A), Candida albicans, Cryptococ cus neoformans, and Paracoccidioides brasiliensis, and mycelium culture of Aspergillus fumigatus and Fusarium spp.were used for all studies.In addition, to the maintenance of the yeast phase of each fungus, cultures were grown at 37°C in brain heart infusion (Gibco Invitrogen Corporation, California, USA) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco Invitrogen Corporation, California, USA), for H. capsulatum, Saboraud dextrose broth (SDB, BD DIFCO, Becton, Dickinson and Com pany, USA), for C. albicans and C. neoformans, and SDB plus 0.14% L-asparagine and 0.01% thiamine hydrochloride.Mycelium culture of A. fumigatus and Fusarium spp. was maintained at 25°C in SDB medium.
The fungal suspension was pelleted by centrifugation (1,500 rpm, 10°C for 10 min), washed two times with PBS (Gibco, Invitrogen Corporation, California, USA), and lysed using liquid nitrogen.Then, the frozen samples were resuspended in lysis buffer [50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% NP-40, and 1× protease inhibitor cocktail (Roche Holding AG, Basel, Switzerland)] and sonicated for 10 min.Finally, cell debris was pelleted by centrifugation at 5,000 rpm for 5 min, resuspended in minimum volume with lysis buffer, and stored at −20°C for subsequent analysis.

SDS-polyacrylamide gel electrophoresis
Purified protein (Hc_Ags), culture extracts of fungi, and urine samples from patients with confirmed histoplasmosis were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).Briefly, each sample was mixed with 2× loading buffer [100 mM Tris-HCL (pH 6.8), 4% (wt/vol) SDS, 20% glycerol, 200 mM β-mercaptoethanol] and boiled for 5 min.The samples were electrophoresed on a 12% SDS-polyacrylamide gel for 90 min at 100 V at room temperature on a Mini-PROTEAN Electrophoresis system (Bio-Rad, San Diego, USA).For visualization of separated proteins, gels were stained with Coomassie blue staining buffer for 1 h, followed by washing with decoloration buffer (10% acetic acid and 5% methanol).Coomassie-stained gels were analyzed and visualized using a Trans illuminator (Molecular Imager Gel Doc XR, Bio-Rad).mPAGE Color Protein Standard (Millipore, Sigma-Aldrich, St. Louis, MO, USA) with a mixture of 10 pre-stained proteins (10-203 kDa) was used as standard size proteins in SDS-PAGE.

Immunoblotting analysis
Immunoblot analysis was performed to evaluate the seroreactivity of serum from mice previously immunized.Briefly, the proteins resolved in SDS-PAGE were transferred onto a polyvinylidene difluoride membrane at 80 V for 90 min using the Trans-Blot Blotting System (Bio-Rad, Hercules, CA, USA).The membrane was washed twice with Tris-buffered saline (TBS) supplemented with 0.1% Tween-20, 1% methanol, blocked for 1 h with 1% skim milk in TBS, and followed by washing three times for 5 min.The membrane was probed with polyclonal serum diluted to 1:1,000 (pooled serum from immunized animals) for 2 h at room temperature by gentle shaking to perform immunoblotting.As a control, commercial monoclonal antibody anti-C.albicans or serum anti-Aspergil lus from anonymous patients with confirmed invasive aspergillosis were used against fungal extracts (Santa Cruz Biotechnology, Texas, USA).Then, the membrane was washed three times and incubated with secondary antibody horseradish-peroxidase-labeled goat anti-IgG mouse antibody (Abcam, Cambridge, UK), dilution 1:10,000) for 1 h at room temperature.Next, the membrane was washed and subjected to chemilumines cence using 3,3′-diaminobenzidine according to the manufacturer's specifications.Band intensity was analyzed by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).mPAGE Color Protein Standard (Millipore, Sigma-Aldrich, St. Louis, MO, USA) with a mixture of 10 pre-stained proteins (10-203 kDa) was used as standard size proteins in western blotting.

FIG 1
FIG 1 Descriptive analysis of OrthoMCL clusters.A non-statistical cladogram is collapsed, showing the relationships and taxonomic classification among the fungal species included in the analysis.The number indicates the phylum; 1: Ascomycota, 2: Basidiomycota, and 3: Actinobacteria.On the right-hide side, the table compares the number of gene families identified by OrthoMCL for each species.

FIG 3
FIG 3 Protein expression and reactivity of PAbs anti-Hc670_Ag and anti-Hc212_Ag.SDS-PAGE (A and C) and western blotting (B and D) of Hc670_Ag (A and B) or Hc212_Ag (C and D) purified proteins, and culture extract collected from H. capsulatum yeast (HcYE) running at 12% with protein size standards molecular weight markers.Same HcYE and pre-stained protein ladder were used for analysis and running of SDS-PAGE.Specific reactivity of PAb of (B) anti-Hc670_Ag or (D) anti-Hc212_Ag obtained from mice immunized was evaluated.

FIG 4
FIG 4 Immunoreactivity of PAbs anti-Hc670_Ag and anti-Hc212_Ag to culture extracts of C. albicans, A. fumigatus, P. brasiliensis, C. neoformans, and Fusarium spp.SDS-PAGE (A) and western blotting (B-E) of culture extracts of C. albicans (Ca), A. fumigatus (Af), Cryptococcus neoformans (Cn), Paracoccidioides brasiliensis (Pb), and Fusarium spp.(Fsp) running at 12% with molecular weight markers.Same HcYE and pre-stained protein ladder were used for analysis and running of SDS-PAGE.Specific reactivity of PAb of anti-Hc670_Ag (B), anti-Hc212_Ag (C), or commercial monoclonal antibody anti-C.albicans (D) and serum anti-Aspergillus (E) were evaluated.

FIG 5
FIG 5 Immunoreactivity of PAbs anti-Hc670_Ag and anti-Hc212_Ag to samples from patients with confirmed histoplasmosis.Immunoblots of individual urine samples from patients with a confirmed diagnosis of histoplasmosis (Hc + #1 to #4) demonstrated reactivity to PAbs anti-Hc670_Ag (A) and anti-Hc212_Ag (B).Purified proteins were used as a control.

TABLE 1
Total number of genes per cluster

TABLE 2
Description of potential diagnostic biomarkers for histoplasmosis (36)me defined by authors.bMolecularweight based on the amino acid sequence.cGeneexpression calculated as fragments per kilobase of exon per million fragments mapped (FPKM).Value as yeast:mycelia ratio (32).dHistoplasma-specificpeptides identified by Crockett et al.(36).